Cholangiocarcinoma (CCA), a malignancy arising from bile duct epithelium, and pancreatic carcinoma are the most common tumor types found in the pancreatobiliary tract. High mortality is associated with these cancers with a five-year survival rate of less than 5%. Pancreatic carcinoma was the tenth most common type of cancer in the United States in 2009 but was the fourth most common cause of cancer death among Americans.
While CCA is a relatively rare tumor, its incidence is increasing worldwide for reasons that are unclear. Evidence suggests that chronic inflammation predisposes biliary epithelium to the development of carcinoma. Therefore, patients with primary sclerosing cholangitis (PSC) are at increased risk of cholangiocarcinoma. PSC patients typically receive regular ERCP surveillance including cytologic brushing of suspicious areas.
Patients with pancreatobiliary malignancy tend to present with symptoms at late stage when chance of successful intervention is low. Early detection offers patients their best chance for surgical resection, which is currently the most viable treatment option.
Acquisition of adequate and diagnostic tissue during endoscopic retrograde cholangiopancreatography (ERCP) is challenging. Successful biopsy collection can be hampered by challenging anatomical location of some structures. Pancreatobiliary biopsies tend to be small, inadequate, and/or lacking diagnostic cells. Pathological evidence of tumor (e.g., biopsy, cytological brushing, and fine needle aspiration (FNA)) is necessary to confirm a suspicion of carcinoma; however, current diagnostic assays have limited sensitivity for detection of malignancy. Cytologic brushings can be performed during ERCP and, because they can sample the entire area under scrutiny for carcinoma, may provide a diagnosis of malignancy when histology is negative, but the sensitivity of routine cytology is suboptimal. The detection rate for carcinoma by routine cytological analysis of ERCP brushings is relatively low (20% at the Mayo Clinic). Use of digital image analysis (DIA) and fluorescence in situ hybridization (FISH) on cytological brushings have improved detection rates for carcinoma over routine cytology; however, DIA has been shown to not be an independent predictor of malignancy and has been discontinued (Fritcher et al., Gastroeneterology 136(7): 2180-2186 (2009)). FISH utilizing probes originally designed for the detection of bladder cancer (UroVysion™, Abbott Molecular, Des Plaines, Ill.) has been shown to be an independent predictor of malignancy (Fritcher et al. (2009), supra). However, while FISH detects approximately twice as many cases of carcinoma compared to routine cytology, it fails to detect half of the cancer specimens. UroVysion™ contains a locus-specific probe directed to the P16 gene, which is a tumor-suppressor gene located on the short arm of chromosome 9 and is known to be deleted in a proportion of pancreatobiliary carcinomas (Caldas et al., Nature Genetics 8(1): 27-32 (1994); see, also, erratum in Nature Genetics 8(4): 410 (1994)); however, a threshold for positivity has not been established and, therefore, to Applicants' knowledge, this probe is not being evaluated in current clinical practice. Imaging modalities, such as computed tomography, ultrasound, and magnetic resonance imaging (MRI), allow for visualization of the pancreatobiliary tract. Differentiation between benign and malignant structures using imaging is not always possible, however. Serum tumor marker levels, such as CA19-9, can be elevated in patients with pancreatobiliary malignancy, but such markers are not specific for carcinoma and, hence, have limited utility.
Miyamoto et al. (Oncology 56(1): 73-82 (1999)) reportedly investigated the expression of several members of the Bcl-2 family proteins in pancreatic cancer using immunohistochemical analysis of 30 invasive ductal adenocarcinomas and 23 intraductal papillary-mucinous tumors (IPMTs) and immunoblot analysis of six cancer tissues and seven pancreatic cancer cell lines. Mcl-1 (myeloid cell leukemia sequence 1) expression was reportedly found in 90% of the invasive ductal adenocarcinomas and 88% of intraductal papillary-mucinous adenocarcinomas. Mcl-1 protein levels were reportedly uniformly high in all pancreatic cancer cell lines.
McKay et al. (HPB (Oxford) 13(5): 309-319 (May 2011; epub Mar. 10, 2011)) reportedly used array comparative genomic hybridization to identify novel potential therapeutic targets in CCA. Regions covering the EGFR (epidermal growth factor receptor) gene, among others, were reportedly frequently gained. Harder et al. (World J. Gastroenterol. 15(36): 4511-4517 (Sep. 28 2009)) have reported that EGFR may be a promising therapeutic target in patients with advanced biliary tract cancer. Yoshikawa et al. (Br. J. Cancer 98(2): 418-425 (Jan. 29, 2008; epub Dec. 18, 2007)) reportedly assessed 236 cases of CCA retrospectively using immunohistochemical analysis of EGFR, among other genes. They reportedly observed an association between over-expression of EGFR with macroscopic type, lymph node metastasis, tumor stage, lymphatic vessel invasion, and perineural invasion in extrahepatic cholangiocarcinoma (EHCC). EGFR expression was reportedly found to be a significant prognostic factor and a risk factor for tumor recurrence. Based on such results, they suggested that EGFR expression is associated with tumor expression in CCA (see, also, Ito et al., Pathol. Res. Pract. 197(2): 95-100 (2001)). Gwak et al. (J. Cancer Res. Clin. Oncol. 131(10): 649-652 (October 2005; epub Oct. 20, 2005)) reportedly observed deletions in exon 19 of the EGFR gene were more commonly found in intra-hepatic or poorly differentiated CCAs.
A gain/amplification of the MYC gene, among others, in pancreatic adenocarcinoma has been reported by Birnbaum et al. (Genes Chromosomes Cancer 50(6): 456-465 (June 2011; epub Mar. 15, 2011)).
Alteration of the P16 gene, among other genes, reportedly is frequently observed in pancreatic ductal adenocarcinoma (PDAC) (Delpu et al., Curr. Genomics 12(1): 15-24 (March 2011)) and intraductal papillary mucinous neoplasm (IPMN) (Huo et al., Zhonghua Bing Li Xue Za Zhi 37(10): 670-675 (October 2008)). Karamitopoulou et al. also disclose that P16 reportedly is an important prognostic marker in CCA (Am. J. Clin. Pathol. 130(5): 780-786 (November 2008)). Tonini et al. disclose that there reportedly is strong evidence for P16, among others, as an independent predictor of patient outcome for pancreatic cancer (Expert Opin. Ther. Targets 11(12): 1553-1569 (December 2007)). Such changes reportedly arise gradually during carcinogenesis. Frequent somatic mutations and homozygous deletion of the P16 (MTS1) gene have been reported in pancreatic adenocarcinoma (Caldas et al. (1994), supra). Others report that such changes can be used to differentiate between cancer and chronic inflammation (see, e.g., Thosani et al., Pancreas 39(8): 1129-1133 (November 2010)).
In view of the foregoing, there remains a need for more sensitive, more reliable, and more informative diagnostic and prognostic methods in the management of pancreatobiliary cancer. The present disclosure seeks to provide a set of markers, as well as methods of use and a kit comprising the set of markers, for the diagnosis, prognosis, and the assessment of the therapeutic or prophylactic treatment of pancreatobiliary cancer. Extensive testing of various markers, as described herein, was necessary to determine the best combination of markers for optimal detection of pancreatobiliary cancer. This and other objects and advantages, as well as inventive features, will become apparent from the detailed description provided herein.